生長因子緩釋微球修復壓力性尿失禁鼠功能及結構的研究
發(fā)布時間：2018-09-13 21:46
【摘要】：背景胰島素樣生長因子-1可用于治療經(jīng)陰道分娩的壓力性尿失禁病人損傷的尿道外括約肌。新的藥物控制釋放系統(tǒng)—海藻酸鈣-多聚鳥氨酸-明膠微球可用于包裹并保持生長因子的活性,實現(xiàn)活性藥物緩慢釋放,持續(xù)發(fā)揮治療作用。目的1.制備IGF-1緩釋微球并檢測其理化性質(zhì),分析微球的緩釋效果;2.構建產(chǎn)傷動物模型,通過功能學試驗以及組織學檢測驗證IGF-1緩釋微球?qū)ζ淠蚩毓δ芗敖Y構的恢復作用。方法1通過乳化交聯(lián)法制備可緩釋IGF-1的海藻酸鈣-多聚鳥氨酸-明膠微球。在光鏡下觀察微球的形態(tài)并進行粒徑分析,對微球的常溫體外孵育的穩(wěn)定性、體外機械強度、生物安全性進行檢測分析,后通過ELISA試劑盒計算微球中IGF-1的包封率、載藥率及釋放特性。2使用陰道擴張來模擬產(chǎn)傷模型。將動物分為四個組,分別是假手術組、生理鹽水組、空載微球組和IGF-1微球組,按分組進行處理。手術一周后進行漏尿點壓試驗和尿道外括約肌肌電圖監(jiān)測。功能檢測后處死,取出尿道組織,處理后切片進行染色,觀察尿道及周圍組織顯微形態(tài)結構改變。結果1微球的制備、理化性質(zhì)及緩釋效果:經(jīng)乳化交聯(lián)法可得到大小均一且穩(wěn)定的微球,平均直徑為(91.72±3.51)μm;微球體外孵育實驗證明微球在等滲透壓生理鹽水中形態(tài)穩(wěn)定,并無膨脹或破裂;微球體外機械強度測試中,震蕩搖晃60小時后,微球的完整率仍為98.4±0.3%,表現(xiàn)出良好的穩(wěn)定性;三個不同濃度(100%、50%、20%)的微球浸出液通過相對增值率RGR計算其細胞毒性分級均為0到1級,證明微球沒有細胞生物毒性;微球釋放IGF-1有“突釋”的特點,累計36小時微球中約90%的IGF-1生長因子被釋放,其后12天內(nèi)的釋放曲線呈現(xiàn)緩慢微量的線性增加趨勢;緩釋微球包裹IGF-1的載藥量為:606.52ng/g,包封率為45.3%。2 IGF-1緩釋微球注射治療女性壓力性尿失禁模型鼠的作用及機制探討:手術一周后對SD鼠進行漏尿點壓試驗,假手術組的漏尿點壓為44.4 ± 3.4 cmH2O,而生理鹽水組與空載微球組的漏尿點壓分別為23.9± 1.3 cmH2O和21.7± 0.8 cmH20,與假手術組相比顯著降低(p0.05),而IGF-1微球組的漏尿點壓為28.4 ± 1.2 cmH2O,比空載微球組有顯著性地提高(p0.05);Masson's Trichrome染色可發(fā)現(xiàn)陰道擴張損傷后的尿道外括約肌有廣泛的斷裂,肌肉排列紊亂,組織間有膠原纖維沉積,經(jīng)過了 IGF-1微球治療后可觀察到肌肉的層數(shù)比生理鹽水組和空載微球組有明顯的恢復,而且膠原纖維的沉積也更少,尿道外括約肌的形態(tài)結構也完整;對于假手術組,CD31陽性的毛細血管密度在生理鹽水組和空載微球組明顯下降,而經(jīng)過IGF-1微球治療的大鼠的尿道組織具有更高密度的毛細血管密度,與生理鹽水組和空載微球組比較有統(tǒng)計學差異(p0.05)。結論使用乳化交聯(lián)法可構建大小均一、形態(tài)穩(wěn)定、有相當機械強度和無生物毒性的海藻酸鈣-多聚鳥氨酸-明膠微球,微球?qū)GF-1的載藥率和釋放特性均符合針對壓力性尿失禁治療需求的生長因子緩釋系統(tǒng)的設計要求;而使用IGF-1緩釋微球治療壓力性尿失禁大鼠能有效促進受損的尿道括約肌的損傷后恢復以及血管再生,能明顯提高大鼠的尿控功能以及改善組織結構。IGF-1緩釋微球有望作為一種新型的治療壓力性尿失禁的方法。
[Abstract]:BACKGROUND Insulin-like growth factor-1 (IGF-1) can be used to treat urethral external sphincter injury in patients with stress urinary incontinence during vaginal delivery. 1. Preparation of sustained-release microspheres of IGF-1 and its physicochemical properties were tested to analyze the sustained-release effect of the microspheres; 2. Establishment of an animal model of birth injury and verification of the recovery of urinary control function and structure of the sustained-release microspheres of IGF-1 by functional test and histological examination. Methods 1. The sustained-release of IGF-1 was prepared by emulsifying and crosslinking method. Microspheres. The morphology of microspheres was observed under light microscope and the size of microspheres was analyzed. The stability, mechanical strength and biological safety of microspheres incubated at room temperature in vitro were tested and analyzed. The encapsulation efficiency, drug loading rate and release characteristics of IGF-1 in microspheres were calculated by ELISA kit. One week after operation, leak point pressure test and external urethral sphincter electromyography (ESEMG) were performed. The urethral tissues were removed and stained. Results 1. Preparation, physicochemical properties and sustained-release effect of the microspheres: The microspheres with uniform size and stable average diameter (91.72 65507 The complete rate of the microspheres was 98.4 6550 The release curve of long-acting factor showed a slow linear increasing trend within 12 days after the release of long-acting factor; the drug loading of sustained-release microspheres encapsulated with IGF-1 was 606.52 ng/g, and the encapsulation rate was 45.3%. 2 The effect and mechanism of sustained-release microspheres on female stress urinary incontinence model rats: leak point pressure test and artificial hand test were carried out one week after operation. The leak point pressure of the operation group was 44.4 (+ 3.4) cmH2O, while that of the saline group and the empty microsphere group were 23.9 (+ 1.3) cmH2O and 21.7 (+ 0.8) cmH20, which were significantly lower than that of the sham operation group (p0.05), while that of the IGF-1 microsphere group was 28.4 (+ 1.2) cmH2O, which was significantly higher than that of the empty microsphere group (p0.05). Staining revealed extensive rupture of the external urethral sphincter after vaginal dilatation injury, disorder of muscle arrangement and deposition of collagen fibers between tissues. In sham operation group, the capillary density of CD31 positive group decreased significantly in normal saline group and no-loaded microsphere group, while the urethral tissue of rats treated with IGF-1 microsphere had higher density of capillary density, which was significantly different from that of normal saline group and no-loaded microsphere group (p0.05). Conclusion Emulsion intercourse was used. The microspheres with uniform size, stable morphology, considerable mechanical strength and non-toxicity could be constructed. The drug loading rate and release characteristics of the microspheres met the design requirements of growth factor sustained-release system for stress urinary incontinence treatment. The sustained-release microspheres of IGF-1 were used to treat stress urine. Incontinence rats can effectively promote the recovery of injured urethral sphincter and vascular regeneration, significantly improve the urinary control function and improve the tissue structure of rats. IGF-1 sustained-release microspheres are expected to be a new treatment for stress urinary incontinence.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R694.54
，
本文編號：2241832
[Abstract]:BACKGROUND Insulin-like growth factor-1 (IGF-1) can be used to treat urethral external sphincter injury in patients with stress urinary incontinence during vaginal delivery. 1. Preparation of sustained-release microspheres of IGF-1 and its physicochemical properties were tested to analyze the sustained-release effect of the microspheres; 2. Establishment of an animal model of birth injury and verification of the recovery of urinary control function and structure of the sustained-release microspheres of IGF-1 by functional test and histological examination. Methods 1. The sustained-release of IGF-1 was prepared by emulsifying and crosslinking method. Microspheres. The morphology of microspheres was observed under light microscope and the size of microspheres was analyzed. The stability, mechanical strength and biological safety of microspheres incubated at room temperature in vitro were tested and analyzed. The encapsulation efficiency, drug loading rate and release characteristics of IGF-1 in microspheres were calculated by ELISA kit. One week after operation, leak point pressure test and external urethral sphincter electromyography (ESEMG) were performed. The urethral tissues were removed and stained. Results 1. Preparation, physicochemical properties and sustained-release effect of the microspheres: The microspheres with uniform size and stable average diameter (91.72 65507 The complete rate of the microspheres was 98.4 6550 The release curve of long-acting factor showed a slow linear increasing trend within 12 days after the release of long-acting factor; the drug loading of sustained-release microspheres encapsulated with IGF-1 was 606.52 ng/g, and the encapsulation rate was 45.3%. 2 The effect and mechanism of sustained-release microspheres on female stress urinary incontinence model rats: leak point pressure test and artificial hand test were carried out one week after operation. The leak point pressure of the operation group was 44.4 (+ 3.4) cmH2O, while that of the saline group and the empty microsphere group were 23.9 (+ 1.3) cmH2O and 21.7 (+ 0.8) cmH20, which were significantly lower than that of the sham operation group (p0.05), while that of the IGF-1 microsphere group was 28.4 (+ 1.2) cmH2O, which was significantly higher than that of the empty microsphere group (p0.05). Staining revealed extensive rupture of the external urethral sphincter after vaginal dilatation injury, disorder of muscle arrangement and deposition of collagen fibers between tissues. In sham operation group, the capillary density of CD31 positive group decreased significantly in normal saline group and no-loaded microsphere group, while the urethral tissue of rats treated with IGF-1 microsphere had higher density of capillary density, which was significantly different from that of normal saline group and no-loaded microsphere group (p0.05). Conclusion Emulsion intercourse was used. The microspheres with uniform size, stable morphology, considerable mechanical strength and non-toxicity could be constructed. The drug loading rate and release characteristics of the microspheres met the design requirements of growth factor sustained-release system for stress urinary incontinence treatment. The sustained-release microspheres of IGF-1 were used to treat stress urine. Incontinence rats can effectively promote the recovery of injured urethral sphincter and vascular regeneration, significantly improve the urinary control function and improve the tissue structure of rats. IGF-1 sustained-release microspheres are expected to be a new treatment for stress urinary incontinence.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R694.54
，
本文編號：2241832
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